The goal of this proposal is to identify those protein components which are involved in regulation of cytoskeletal function in normal and transformed cells. Specifically, we will be isolating and characterizing proteins that are uniquely associated with the fibrous cytoskeletal elements, the actin filaments, microtubules and 100 A filaments. There is now abundant evidence showing that the cytoskeletal elements are altered with the shape change accompanying transformation in many cells and we believe that the secondary proteins associated with the fibrous cytoskeletal elements may help to regulate their organization in both normal and transformed cells. Our experimental methods have been especially designed so that we may examine the proteins associated with intact cytoskeletal elements in order to avoid many of the artifacts attendant with methods involving polymerization and depolymerization of cytoskeletal elements. In this approach we will use cytoskeletal stabilizing buffers in combination with nonionic detergents in order to extract soluble proteins from the cells while leaving cytoskeletal elements with their associated proteins intact. A powerful two dimensional gel electrophoresis system developed in our laboratory will be used for protein analysis of cell cytoskeletal models. This method, in combination with radioactive labels and autoradiography allows extremely sensitive detection of very minor protein components. Additional isolation methods will involve the use of affinity binding techniques to selectively extract cytoskeletal associated proteins from cell homogenates. These methods will be applied to several lines of normal and transformed cells in culture and to normal and malignant tissue. Experimental procedures which alter the cytoskeleton in certain cell lines (cyclic AMP treatment) will also be assayed for any effects upon the cytoskeletal associate proteins.